Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins This competitive revision responds to the following NIH program: the Notice Number (NOT- OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The research project focuses on the development of efficient chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is a major posttranslational modification of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. The covalently linked oligosaccharides can profoundly affect proteins'structure, function, and their serum half-life. However, a major challenge in glycobiology research is to deal with the structural heterogeneity of glycoproteins, which are typically produced as mixtures of various glycoforms. Since pure glycoforms are extremely difficult to isolate from natural and recombinant sources, synthesis has become an essential source for obtaining homogeneous materials. The parent grant aims to explore the transglycosylation potential of endo-2-N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for constructing N-glycopeptides and N-glycoproteins. It focuses on studies of substrate specificity, model studies with ribonuclease B, and preliminary application for human IgG1-Fc glycosylation engineering. Our recent discovery of novel ENGase-based glycosynthases for the construction of homogeneous natural N-glycoproteins represents a major advance in the area that has not been proposed in the parent proposal. In addition, the recently solved crystal structure of Endo-A has provided exciting new opportunity to engineer the enzyme for enhancing the transglycosylation efficiency and for broadening its substrate specificity. This competitive revision application intends to expand the scope of the chemoenzymatic method and to speed up its application for constructing biologically important glycoproteins for structural and functional studies. It has two specific aims: 1) Site-directed mutagenesis of ENGases to enhance the efficiency and to expand the scope of chemoenzymatic method;2) chemoenzymatic synthesis of pure glycoforms of selected glycoproteins for structural and functional studies. These will include specific glycoforms of human CD2 extracellular domain for ER-associated degradation (ERAD) studies, and pure glycoforms of ribonuclease B and CD2 for NMR structural studies. This revision application significantly expands the scope and direction of the parent grant. PUBLIC HEALTH RELEVANCE: This competitive revision application aims to develop new methods for the efficient synthesis of glycoproteins for structural and functional studies. The information gained will be valuable for development of glycoprotein-based therapeutics.